The phenylalanine hydroxylase enzyme catalyzes the conversion of the large neutral amino acid phenylalanine (Phe) to tyrosine, which is the rate-limiting step in the catabolism of Phe. The brain is highly sensitive to levels of Phe, and deficiencies in the PAH enzyme may result in excess levels of Phe or hyperphenylalanemia. Deficiencies in PAH enzyme activity may range from classical PKU and its potential for severe central nervous system dysfunction (mental retardation), to moderate elevations in plasma Phe with no known clinical consequences. A deficiency in PAH enzyme activity is the most common cause of hyperphenylalanemia, with 99% of the mutant alleles mapping to the PAH gene and the remainder mapping to several genes involved in the synthesis and recycling of tetrahydrobiopterin (BH.sub.4), the cofactor in the hydroxylation reaction (Scriver, C. R., "Whatever happened to PKU?", Clin. Biochem., 1995, 28: 137-144). The human PAH gene spans 90 kb, is comprised of 13 exons, and has been localized to chromosome 12q24.1 (Lidsky, A. S. et al., "Regional mapping of thephenylalanine hydroxylase gene and the phenylketonuria locus in the human genome", Proc Natl Acad Sci USA, 1985, 82:6221-6225; DiLella, A. G. et al., "Molecular structure and polymorph map of the human phenylalanine hydroxylase gene", Biochemistry, 1986, 25:743-749). Structure/function analyses have identified a central catalytic domain which contains sites for substrate, iron and BH.sub.4 cofactor binding, an N-terminal region with regulatory properties, and a C-terminal domain involved with intersubunit binding (Hufton, S. E. et al., "Structure and function of the aromatic amino acid hydroxylases", Biochem J, 1995, 311:353-366; Waters, P. J. et al., "In vitro expression analysis of mutations in phenylalanine hydroxylase: linking genotype to phenotype and structure to function", Hum Mutat, 1998, 11:4-17).
Although some studies have suggested that patients with PKU and heterozygotes for PKU might have an increased susceptibility for psychiatric disorders such as schizophrenia (Thompson, J. H., "Relatives of phenylketonuric patients", J. Ment. Defic. Res. 1957; 1:67-78; Kuznersova, L. I., "Frequency and phenotypic manifestations of schizophrenia in the patients with phenylketonuria", Sov. Genet. 1974:554-555) this remains a topic of considerable controversy. Several investigations have found that there is no statistical evidence to demonstrate that the frequency of mental illness in PKU patients and their families exceeds levels in control populations (Perry, T. L. et al., "The incidence of mental illness in relatives of individuals suffering from phenylketonuria or mongolism", J. Psychiatr. Res. 1966; 4:51-57; Blumenthal, M. D., "Mental illness in parents of phenylketonuric children", J. Psychiatr. Res. 1967; 5:59-74; Larson, C. A. and Nyman, G. E., "Phenylketonuria: mental illness in heterozygotes", Psychiatr. Clin. 1968; 1:367-374; Pietz, J. et al., "Psychiatric disorders in adult patients with early-treated phenylketonuria", Pediatrics, 1997, 99:345-350).
Investigations of Phe metabolism in psychiatric disorders are few and the findings are equivocal. Some studies have indicated that schizophrenic patients have higher levels of plasma Phe than control subjects (Poisner, A. M., "Serum phenylalanine in schizophrenia; biochemical genetic aspects", J. Nerv. Ment. Dis. 131:74-76, 1960; Bjerkenstedt et al., "Plasma amino acids in relation to cerebrospinal fluid monoamine metabolites in schizophrenic patients and healthy controls", Br. J. Psychiatry, 1985, 147:276-282; Rao, M. L. et al., "Serum amino acids, central monoamines, and hormones in drug-naive, drug-free, and neuroleptic-treated schizophrenic patients and healthy subjects", Psychiatry Res. 1990, 34:243-257), while another reported no significant difference between the two groups (Szymanski, H. V. et al., "Plasma phenylethylamine and phenylalanine in chronic schizophrenic patients", Biol. Psychiatry 1987; 22:194-198). The results for another index of Phe metabolism (the level of plasma Phe following a Phe challenge) have been similarly ambiguous, with a report from one group of investigators that post-challenge Phe levels were increased in schizophrenic patients compared to controls, which they subsequently failed to replicate (Wyatt, R. J. et al., "Phenylethylmine (PEA) and chronic schizophrenia", Catecholamines: Basic and Clinical Frontiers, Usdin, K., Kopin, I. J., Barchas, J. D., eds. New York: Raven Press, 1979; 1833-1835; Potkin, S. G. et al., "Plasma phenylalanine, tyrosine, and tryptophan in schizophrenia" Arch. Gen. Psychiatry 1983; 40: 749-752).
Another aspect of the studies on Phe metabolism in schizophrenia have focused on phenylethylamine, which is produced in excess by a decarboxylase pathway in the absence or impairment of PAH activity. Phenylethylamine has been proposed to act as a neuromodulator via serotonin receptors (Boulton, A. A. et al., "Phenylethylamine in the CNS: effects of monoamine oxidase inhibiting drugs, deuterium substitution and leisons and its role in the neuromodulation of catecholaminergic neurotransmission", J. Neural. Transmission Suppl. 1990; 29: 119-129, Sloviter, R. S. et al., "Serotonergic properties of b-phenethylamine in rats", Neuropharmacology 1981; 20: 1067-1072) and is of interest in schizophrenia research since it is a chemical congener of amphetamine, abuse of which produces a psychosis resembling paranoid schizophrenia (Snyder, S. R., "Amphetamine psychosis: a "model" schizophrenia mediated by catecholamines", Am. J. Psychiatry 1973; 130: 61-67). Investigators have reported differences between schizophrenic patients and control subjects including differences between groups of schizophrenic subtypes, as measured by the level of phenylethylamine in body fluids (Szymanski, H. V. et al., "Plasma phenylethylamine and phenylalanine in chronic schizophrenic patients", Biol. Psychiatry 1987; 22: 194-198.69; Potkin, S. G. et al., "Phenylethylamine (PEA) and phenylacetic acid (PAA) in the urine of chronic schizophrenic patients and controls", Psychopharmacol. Bull. 1980; 16:52-54; Jeste, D. V. et al., "Cross-cultural study of a biochemical abnormality in paranoid schizophrenia", Psychiatry Res. 1981; 5: 341-352; Yoshimoto, S. et al., "Urinary trace amine excretion and platelet monoamine oxidase activity in schizophrenia", Psychiatry Res. 1897; 21:229-236; O'Reilly, R. et al., "Plasma phenylethylamine in schizophrenic patients", Biol. Psychiatry 1991; 30:145-150; Fischer, E. et al., "Urinary elimination of phenethylamine", Biol Psychiatry 1972; 5: 139-147). These studies on phenylethylamine, though more numerous than those on Phe, have been considered particularly problematic because of the trace amine status of phenylethylamine (small amounts, rapid transit through systems, unstable) which has resulted in technological difficulties in measurement of the amino and lack of conformity in values across studies.
The study of Phe metabolism is the most limited in psychiatric disorders other than schizophrenia. In patients with endogenous depression, unipolar depression or bipolar syndrome, the response to a Phe challenge was indistinguishable from that in control subjects (Pratt, R. T. C. et al., "Phenylalanine tolerance in endogenous depression", Brit. J. Psychiat., 1963, 109:624-628; Targum, S. D. et al., "Screening for PKU heterozygosity in bipolar affectively ill patients", Biol. Psych., 1979, 14:651-655; Gardos, G. et al., "The acute effects of a loading dose of phenylalanine in unipolar depressed patients with and without tardive dyskinesia", Neuropsychopharmacology, 1992, 6:241-247).
Only two investigations have examined the PAH gene and schizophrenia and both have produced negative findings. The first of these screened for two specific mutations in the PAH gene (the putative null mutations R408W and IVS 12nt1) in schizophrenics and normal controls. None of the schizophrenic subjects in the study were found to have these variants. It was thus concluded that neither of these genetic polymorphisms were associated with a predisposition to schizophrenia (Sobell, J. L. et al., "Novel association approach for determining the genetic predisposition to schizophrenia: case-control resource and testing of a candidate gene", Am. J. Med. Genet. 1993; 48:28-35). The second study was a genome-wide search for schizophrenia susceptibility genes. The investigators failed to find a significant association between PAH and disease susceptibility (Shaw, S. H. et al., "A genome-wide search for schizophrenia susceptibility genes", Am. J. Med. Genet. 1998; 81:364-376).
No other studies have found an association between the PAH locus on chromosome 12 in reference to psychiatric disorders. One publication describes an investigation of the relationship between schizophrenia and the phospholipase-A2 gene, the result of which was negative (Psychiatr Genet. 1995, 5:177-80; O-Malley, M. P. et al. Linkage analysis between schizophrenia and the Darier's disease regionon 12q, Psychiatr Genet. 1996: 6:187-90).
The psychotic disorders are the most serious of the psychiatric illnesses and make up the bulk of the patients in the public sector extracting a major economic cost from society. Patients afflicted with psychosis suffer from symptoms such as delusions, hallucinations, disorganized speech and grossly disorganized or catatonic behavior. The disorders in this category are schizophrenia, schizophreniform disorder, schizoaffective disorder, brief psychotic disorder, delusional disorder, shared psychotic disorder, psychotic disorder due to a general medical condition, substance-induced psychotic disorder, psychotic disorder not otherwise specified (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Washington, D.C., American Psychiatric Association, 1994).
The most prevalent of these disorders is schizophrenia having a lifetime prevalence ranging from 0.5% to 1%, the particular symptoms of which are the following (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Washington, D.C., American Psychiatric Association, 1994):
1. Delusions--are erroneous beliefs that usually involve a misinterpretation of perceptions or experiences. Presecutory delusions are the most common; the person believes he or she is being tormented, followed, tricked, spied on, or subjected to ridicule. Referential delusions are also common; the person believes that certain gestures, comments, passages from books, newspapers, song lyrics or other environmental cues are specifically directed at him or her. Bizarre delusions are especially characteristic of schizophrenia. An example of a bizarre delusion is a person's belief that a stranger has removed his or her internal organs and has replaced them with someone else's organs without leaving any wounds or scars.
2. Hallucinations--auditory hallucinations are the most common and are experienced as voices that are perceived as distinct from the person's own thoughts. Pejorative or threatening voices are the most common. Most characteristic of schizophrenia are two or more voices conversing with one another or voices maintaining a running commentary on the person's thoughts or behaviors.
3. Disorganized thinking--formal thought disorder, loosening of associations; as manifested by speech that is disorganized enough to substantially impair effective communication; such as, derailment where the person slips off the track of a thought, answers to questions may be completely unrelated, speech may be so severely disorganized that it is incomprehensible as resembling a word salad.
4. Grossly disorganized behavior--problems may be seen in any form of goal-directed behavior, leading to problems performing activities of daily living. For instance, the person may be markedly disheveled, dress in an unusual manner (e.g., multiple overcoats on a hot day), show inappropriate sexual behavior (e.g., public masturbation), show childlike silliness, show unpredictable and untriggered agitation.
5. Catatonic motor behaviors--a marked decrease of reactivity to the environment which can reach to stupor, maintaining a rigid posture and resisting efforts to be moved, inappropriate or bizarre postures, or purposeless and unstimulated excessive motor activity.
6. Negative symptoms--person's face appears immobile and unresponsive with poor eye contact and reduced body language, speech is characterized by brief, laconic, empty replies, person has an inability to initiate and persist in goal-directed activities, and will sit for long periods of time and show no interest in participating in work or social activities.
The disorders in the category of Mood Disorders are; Major Depressive Disorder, Dysthymic Disorder, Depressive Disorder Not Otherwise Specified, Bipolar I Disorder, Bipolar II Disorder, Cyclothymic Disorder, Bipolar Disorder Not Otherwise Specified, Mood Disorder Due to a General Medical Condition, Substance-Induced Mood Disorder, Mood-Disorder Not Otherwise Specified (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Washington, D.C., American Psychiatric Association, 1994).
The disorders in the category of Personality Disorders are; Paranoid Personality Disorder, Schizoid Personality Disorder, Schizotypal Personality Disorder, Antisocial Personality Disorder, Borderline Personality Disorder, Histrionic Personality Disorder, Narcissistic Personality Disorder, Avoidant Personality Disorder, Dependent Personality Disorder, Obsessive-Compulsive Personality Disorder, Personality Disorder NOS (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Washington, D.C., American Psychiatric Association, 1994).
It was through the results of several independent studies in these patient groups, that the Phe metabolic model was developed and which forms the background of the invention. The model is based on the following established criteria in relation to the role of Phe in neurotransmitter synthesis.
The main factors determining the entry of Phe into the brain are its concentration in plasma, the concentration of the competing large neutral amino acids in plasma, and the activity of the blood-brain barrier transport system or L-system. The L-system, which competitively mediates the bi-directional flux of the neutral amino acids between the blood and brain, has a preferential affinity for the large neutral amino acids, with Phe having the highest affinity among them (Daniel, P. M. et al. "Amino acid precursors of monoamine neurotransmitters and some factors influencing their supply to the brain" Psychol. Med. 1976; 6: 277-286; Pardridge, W. M. and Choi, T. B., "Neutral amino acid transport at the human blood-brain barrier", Federation Proceedings, 1986, 45: 2073-2078). Due to the competition between amino acids for brain entry, higher concentrations of plasma Phe can result in lower brain availability of tyrosine and tryptophan. These latter amino acids serve as precursors in the synthesis of the neurotransmitters dopamine, serotonin and noradrenaline, all of which have been implicated in psychotic, mood and personality disorders. Further, an inhibitory effect of Phe and its metabolites (such as phenylethylamine and phenylacetic acid) on the activities of tyrosine hydroxylase, tryptophan hydroxylase, and DOPA decarboxylase further decreases the synthesis of dopamine, noradrenaline and serotonin (Caballero, B. and Wurtman, R. J., "Control of plasma phenylalanine levels" Ch. 1, In: Wurtman, R. J. and Ritter-Walker, E., eds. Dietary Phenylalanine and Brain Function. Boston: Birkhauser, 1988; 3-12; Lehnert, H. and Wurtman, R. J., "Amino acid control of neurotransmitter synthesis and release: physiological and clinical implications" Psychotherapy & Psychosomatics, 1993; 60: 18-32; Maher, T., "Effects of phenylalanine on the synthesis, release and function of catecholaminergic systems" in Amino Acids in Psychiatric Disease. Washington: American Psychiatric Press, Inc., 1990; 131-142; Scriver, C. R. and Rosenberg, L. E., "Phenylalanine" Ch. 15, In: Schaffer, Al, eds. Amino Acid Metabolism and its Disorders. Philadelphia, 1973; 290-337). Thus the integrity of the Phe metabolic system is critically important in the function of neurotransmitter systems thought to be involved in these disorders. Higher plasma Phe levels have been shown to be significantly associated with lower central nervous system levels of tyrosine and trytophan in a sample of schizophrenics (Bjerkenstedt, L. et al. "Plasma amino acids in relation to cerebrospinal fluid monoamine metabolites in schizophrenic patients and healthy controls" Br. J. Psychiatry, 1985; 147: 276-282).
The Phe metabolic model was initially developed to allow for the development of treatment strategies for a neurological disorder, tardive dyskinesia (TD). This disorder is a side effect of neuroleptic treatment (drugs that block dopamine function, and more recently in the atypical form also block serotonin function) which has been the primary treatment for schizophrenia, and the other psychoses. It was suggested that the symptoms of the disorder were due to a dopaminergic supersensitivity brought on by years of treatment with dopamine-blocking drugs. More recent studies have suggested that lifelong reduced dopamine synthesis due to chronically higher levels of plasma Phe may increase the vulnerability to the development of TD. These drugs are also used in the treatment of mood and personality disorders. Persons with mood disorders have been shown to have a particular vulnerability to the development of TD when treated with neuroleptics. All three groups of these disorders are believed to at least partially respond to treatment with medications that regulate amine neurotransmitter synthesis.
A summary of the studies which form the core of the Phe metabolic model are as follows:
1. The association of hyperphenylalanemia with TD (Study 1). A point prevalence study of TD (n=211) in a mentally retarded population showed that phenylketonuria, a severe hyperphenylalaninemia, was a significant risk factor for the presence of TD. These data suggested that high plasma Phe was associated with the development of TD (Richardson, M. A. et al., "The prevalence of tardive dyskinesia in a mentally retarded population" Psychopharmacol. Bull., 1986; 22: 243-249).
2. Protein challenge given to schizophrenic men with and without TD. A dietary challenge in the form of a high-protein meal (Phe=3.6% of total protein; branched chain amino acids (BCAA=19.6% of total protein) was administered to 53 male schizophrenics (Study 2). The findings were:
(a) Alteration of protein kinetics in men with TD. Data analyses established that the post-challenge plasma Phe level and post-challenge Phe/large neutral amino acid ratio were significantly higher in patients with TD and were significant predictors of TD status in male schizophrenic patients, independent of age. The ratio corrects for the competition of the other large neutral amino acids with Phe at the blood-brain barrier (Richardson, M. A. et al., "The plasma phenylalanine/large neutral amino acid ratio: a risk factor for tardive dyskinesia" Psychopharmacol. Bull., 1989; 25: 47-51; Richardson, M. A. et al., Comment on `The ratio of plasma phenylalanine to other large neutral amino acids is not a risk factor for tardive dyskinesia` J. Psychopharm., 1993; 7: 2; Richardson, M. A. et al., "Tardive dyskinesia and phenylalanine metabolism: risk-factor studies" Ch. 13, in: Yassa, R. et al., eds. Neuroleptic-Induced Movement Disorders. Cambridge: Cambridge University Press, 1997: 161-174; Richardson, M. A. et al., "Plasma phenylalanine: A measure of tardive dyskinesia vulnerability in schizophrenic males" Chap. 7, in: Richardson, M. A., eds. Amino Acids in Psychiatric Disease. Washington, D.C.: American Psychiatric Press, 1990: 143-160).
(b) Unexpected total remission of TD symptoms. More than half of the patients with TD had either a complete remission of their TD symptoms or a minimum 50% decline in symptoms two hours after the protein meal. Plasma analyses demonstrated that there were significantly lower Phe/large neutral amino acid and tyrosine/large neutral amino acid ratios and significantly higher branched chain amino acid/large neutral amino acid ratios in the group of patients who had a remission, as compared with those who did not. The authors hypothesized that the protein challenge comprised of high branched chain amino acid content, may have reduced the availability of Phe and tyrosine to the central nervous system, effecting the dramatic symptom remission (Richardson, M. A. et al., "Phenylalanine to serotonin to tardive dyskinesia: A new model", Proceedings of the 2nd International Symposium on Serotonin, from Cell Biology to Pharmacology and Therapeutics, Houston, Tex. Sep. 15-18 1992; Richardson, M. A. et al., "A dietary intervention decreases tardive dyskinesia symptoms" Am. Psychiatr. Assoc., 149th Annual Meeting, New York, N.Y. 1996; 0:194).
3. Alteration of Phe kinetics in men with TD (Study 3). The study hypothesis, that TD would be associated with significantly higher plasma Phe indices (absolute plasma Phe level, plasma Phe/large neutral amino acid ratio) two hours after a Phe challenge, was verified for the male participants in the study (N=209; total N=312). The altered kinetics of Phe in men with TD indicated that there was a greater availability of Phe to the brains of these men. These data suggest that the disorder may be related to the effects of this greater availability. Such effects could be the direct neurotoxic effects of Phe and its metabolites and/or the modulating effects of these compounds on the synthesis of the monoamine neurotransmitters (Richardson, M. A. et al., "Tardive dyskinesia and phenylalanine metabolism: risk-factor studies". Ch. 13, in: Yassa, R. et al., eds. Neuroleptic-Induced Movement Disorders. Cambridge; Cambridge University Press, 1997: 161-174; Richardson, M. A. et al., "Phenylalanine metabolism: Sex and Age issues" Schizophrenia Research, 1996; 18: 149; Richardson, M. A., Reilly, M. A., Read, L. L., Flynn, C. J., Suckow, R. F., Maher, T. J., Sziraki, I.: Phenylalanine kinetics are associated with tardive dyskinesia in men but not in women. Psychopharmacology, in press).
4. Treatment of TD with a dietary supplement of the BCAA (Study 4). Clinical trials were conducted to determine whether a drink containing exactly the same amount and proportion of the BCAA as was in the protein challenge meal in Study 2 (given three times a day for two weeks) would decrease TD symptoms (Richardson, M. A., Bevans, M. L., Weber, J. B., Gonzalez, J. J., Flynn, C. J., Read, L. L., Suckow, R. F., Maher, T. J.: Branched chain amino acids decrease tardive dyskinesia symptoms. Psychopharmacology, in press; (Richardson, M. A. et al., "A dietary intervention decreases tardive dyskinesia symptoms" Am. Psychiatr. Assoc., 149th Annual Meeting, New York, N.Y. 1996; 0:194; Richardson, M. A. et al., "TD symptom decreases with regulation of plasma large neutral amino acids". Schizophrenia Research 1997; 24:272; Richardson, M. A. et al., "TD symptom decreases with regulation of plasma large neutral amino acids" Abstracts, 16th International Congress of Nutrition, Montreal, Canada 1997; 0:42). A statistically significant decrease in the level of TD symptoms was observed for the sample. The symptom changes were also clinically significant in that 6 of the 9 subjects had symptom decreases of at least 58%, with all subjects having a decrease of at least 38%. Branched chain amino acid administration increased plasma branched chain amino acid concentrations and branched chain amino acid/large neutral amino acid ratios, and decreased plasma levels and large neutral amino acid ratios of Phe, tyrosine, and tryptophan. Analyses indicated a strong significant correlation between the percent increase in the plasma branched chain amino acid values at the first administration and the percent improvement in TD over the trial in eight of the nine subjects. The study findings suggested that the decrease in TD symptoms was modulated by a decrease in the brain uptake of Phe, a decrease in neurotransmitter synthesis, and/or the increase of the branched chain amino acid and decrease of aromatic amino acids in the periphery.
5. Placebo-controlled trial for the BCAA as a treatment for TD in adult men (Study 5). A placebo-controlled trial of the BCAA drink for the treatment of TD in men was designed to eliminate ineffective doses. For a response criteria of 50% decrease in symptoms, no responders were observed at the placebo dose, or at a low dose. A 17% rate of responders was observed at the mid-dose, and a 50% rate of responders in the high dose group. The trend in these data suggested a significant active versus placebo group response.
6. Pilot trial of the BCAA in the treatment of dyskinesias in neuroleptic-treated children and adolescents 9 (Study 6). Substantial decreases in symptoms for dyskinesias were seen in five out of six adolescents treated for two weeks. The decreases ranged from 37% to 68%. Two adolescent boys went into roll-on treatment periods. The percent decrease in symptoms was accelerated in the longer treatment periods. One subject who showed a 50% decrease after two weeks of treatment showed a 94% decrease after 31 days, and a second subject who showed a 54% decrease after two weeks of treatment showed a 74% decrease after 54 days of treatment.
7. DNA analysis of the PAH gene in psychiatric patients and controls (Study 7). The association between TD and diminished Phe kinetics in combination with the reduction of TD symptoms following a medical food product, led to the screening of the PAH gene in patients with psychotic, mood and personality disorders both with and without TD. The study hypothesis was that PAH variants may cause modest but significant changes in PAH activity that result in differences in Phe kinetics following a dietary Phe dose. It was further hypothesized that PAH variants may also have long term effects including predisposition to TD and psychotic disorders. The findings of this study comprise the object of the invention, which is presented in the Field of Invention, and described in the Detailed Description and Examples 1-7.